PROJECT SUMMARY Aortic valve disease is the third most common cardiovascular disease in the United States. Calcific aortic valve disease (CAVD) is by far the most common cause, yet it has no effective pharmacologic therapy. Currently, all theories of pathophysiology focus on the contribution of resident aortic valve interstitial and endothelial cells (AVICs and AVECs respectively). However, macrophages make up a significant fraction of the native aortic valve, and pathology studies and unbiased transcriptomics of the calcified aortic valve have observed enrichment of macrophages and T and B lymphocytes. In addition, transcription factor STAT3 and its key activator IL-6 ?both markers of Th17 inflammation ? have been found to be increased in in vitro and in vivo calcification. Recognizing the interplay of immune cell function with endothelial and cardiovascular biology, we have begun to investigate the contribution of immune cells to CAVD pathophysiology. Our in vitro studies of STAT3 blockade show an abrogation of mechanical and transcript-level characteristics of dystrophic calcification. In addition, culture of aortic valve cells with macrophages augments the calcification phenotype. This background information and preliminary data suggest a mechanism for immune cell contribution to the pathophysiology of CAVD. Specifically, we suspect that known calcification stimuli induce an inflammatory phenotype in hematopoietic cells, leading to T lymphocyte infiltration and STAT3-dependent calcific nodule formation. In Aim 1 we will utilize bone marrow transplants and inhibition of lymphocyte maturation via Rag1 knockout to test the role of hematopoietic cells on the whole and lymphocytes, respectively, in the Notch1+/- mouse model of CAVD. To test the impact of in vitro models of calcification on immune cells, in Aim 2 we will utilize in vitro co-culture models of AVICs and macrophages, and probe the effects on STAT3 signaling and AVIC calcification. Aim 2 will interrogate both the calcification outcomes of valvular cells and the immunological outcomes of macrophages. To test the clinical translation of this model, in Aim 3 we will assess the progression of aortic stenosis in patients taking methotrexate, a drug that inhibits T lymphocyte maturation and thus adaptive immune system activity. With these investigations, this project will (a) be the first to mechanistically assess the impact of immune cells in CAVD, (b) contribute directly to the clinical knowledge base on aortic valve disease, and (c) advance the basic science understanding of immune cell physiology under mechanical strain. Finally, this project will not only advance scientific knowledge on aortic valve disease, but further serve as the foundation of my training to be an independent physician-scientist.